SUMMARY This R01 grant focuses on neurotrophic mechanisms involved in stimulant- or opioid-induced plasticity in the nucleus accumbens (NAc) and other reward-related brain regions. A particular focus has been the molecular basis by which these drugs of abuse alter the density and morphology of dendritic spines elaborated by NAc medium spiny neurons (MSNs). Four key questions are addressed in this competitive renewal application. First, virtually all molecular mechanisms implicated in mediating cocaine-induced spine growth affect thin spines only, with little insight available into the delayed growth of mushroom spines seen after prolonged withdrawal from cocaine self-administration (SA) and associated with relapse. Recent work supported by this grant has uncovered the involvement of RAP1B and microtubule-associated proteins in controlling mushroom spines on NAc MSNs, and these novel pathways will be further delineated in male and female mice in the proposed research. Second, there is very little information available concerning whether the time-dependent effects of cocaine SA on NAc dendritic spines occur in D1-type or D2-type MSNs. We have optimized a method to enable this analysis such that the effects RAP1B and microtubules will be studied in a cell type- specific manner. However, we know that MSNs are far more heterogeneous: a subset of MSNs express D1 and D2 receptors, and single cell RNA sequencing reveals considerably more heterogeneity. In the proposed studies, we will embrace this complexity by characterizing cocaine?s effects on NAc MSNs by use of single cell technology. We will also identify the small subset of MSNs that are activated during the life cycle of cocaine SA by use of Arc-CreER2 mice, which make it possible to tag cells activated in response to one stimulus (e.g., first cocaine exposure) and track how those cells respond over time during drug SA. Characterizing dendritic spine regulation in these distinct subsets of MSNs is a high priority. Third, several forebrain regions send excitatory inputs to NAc MSNs, which contribute to distinct aspects of addiction-related behaviors, but much less is known about the cellular heterogeneity within those afferent regions, and how the distinct subsets of neurons are affected by drugs of abuse. Recent findings revealed distinct subsets of ventral hippocampal (vHIP) pyramidal neurons that express either D1 or D2 dopamine receptors, suggesting a parallel level of cellular organization as elaborated for NAc. We have confirmed these findings and found further that such D1 vs. D2 vHIP neurons, both of which innervate the NAc, exert opposite effects on cocaine relapse behavior. This grant offers the perfect venue in which to provide initial characterization of these cell types in addiction. Fourth, much less is known about the effects of opioids on NAc spine plasticity, a major gap in knowledge which we propose to fill through the course of the proposed studies. Together, this work will contribute to an improved understanding of the molecular, cellular, and circuit basis of addiction-related behavioral abnormalities, and provide new pathways forward in identifying target genes suitable for future drug discovery efforts.